Localization of a pH-dependent, A2 subunit-interactive surface within the factor VIIIa A1 subunit

摘要

Factor VIIIa can be reconstituted from A2 subunit and A1/A3-C1-C2 dimer in a reaction that is facilitated by slightly acidic pH. We recently demonstrated that a truncated A1 (A1(37-336)) possessed markedly reduced affinity for A2 compared with intact A I, but retained 30% of native factor VIIIa activity in the presence of A3-C1-C2. We now identify A1-interactive regions for A2 using A1 fragments derived from a limited cryptic digest. Unfractionated trypsin-cleaved At inhibited reconstituted factor VIIIa activity. Two fragments, designated A1(37-121) and A1(221-136), markedly inhibited factor VIIIa reconstitution with either native A1 (K-i = 340 and 194 nM, respectively) or with A1(37-136) (K-i=69 and 116 nM, respectively) at pH 6.0. A third fragment designated A1(122-206) did not possess inhibitory activity. At pH 7.2, the A1(221-336) partially inhibited reconstitution, whereas the A1(37-121) possessed little if any inhibitory activity. Both fragments inhibited factor VIIIa reconstitution as judged by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled At forms at pH 6.0. Furthermore, covalent cross-linking between A2 and A1(37-121) but not A1(221-336) was observed following reaction with a zero-length crosslinker. These findings demonstrate the presence of an extended, pH-dependent A2-interactive surface within regions 37-121 and 221-336 of A1. This interactive surface appears conformationally labile in the truncated At as judged by its apparent stabilization following association with A3-C1-C2. (C) 2004 Elsevier B.V All rights reserved.